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J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. Characterizing NAD- and NADP-dependent of Strawberries

William C. Mitchell1 and Gojko Jelenkovic Plant Science Department, Rutgers University, New Brunswick, NJ 08903 Additional index words. Fragaria ×ananassa, fruit ripening, , organoleptic quality Abstract. The NAD-dependent and NADP-dependent alcohol dehydrogenase activities of strawberries (Fragaria xanan- assa Duch.) were found to have broad specificities including those and responsible for strawberry aroma and flavor either directly or through their ester products. NAD-dependent activities were greatest against short-chained alcohols, whereas the NADP-dependent activities were most active against aromatic and terpene alcohols. Differences were seen in substrate specificity between receptacle and achene alcohol dehydrogenase activities. Alcohol dehydrogenase activities were found to be developmentally regulated in receptacle tissue and increased during the period of fruit maturation and ripening. Isoelectric focusing of NAD-dependent ADH activities showed that several isozymes of this exist, that they differ between receptacle and achene tissues, and that they vary among specific genotypes. Our results suggest that NAD- and NADP-dependent ADH activities are integral components of flavor and fragrance volatile production in ripening strawberries.

NAD-dependent (NAD-ADH; E.C. 1.1.1.1) and NADP-depen- Materials and Methods dent alcohol [NADP-ADH (cinnamyl alcohol dehydrogenase); E.C. 1.1.1.2] exist in a wide variety of higher Plant material. Primary fruit were freshly harvested from straw- plant tissues (Davies et al., 1973; Freeling and Bennett, 1985; berry plants that were either grown in a greenhouse (genotype NJ Hatanaka et al., 1973; Kanellis et al., 1991; Noueiry, 1989; Roe et 8343-6) or field (NJ 8336-1, NJ 8611-1, ‘Earliglow’, ‘Raritan’). al., 1984; Tress1 and Albrecht, 1986; Yamashita et al., 1978). Four developmental stages of strawberries were used in this Putative roles attributed to plant alcohol dehydrogenases include study. Stage 1 and stage 2 fruit had fully white receptacles and surviving periods of hypoxia (Freeling and Bennett, 1985; Kanellis green achenes. Stage 3 fruit had receptacles with pink and white et al., 1991; Roe et al., 1984), regulation of development (Mitchell sections and green achenes. Stage 4 fruit were fully red, fragrant, and Jelenkovic, 1992; Roe et al., 1984), production of cinnamyl and had brown achenes. Average fresh weights of stage 1 through alcohols for the lignification of cell walls (Davies et al., 1973), stage 4 fruit were 0.315 ± 0.056 g, 1.204 ± 0.106 g, 6.492 ± 1.236 protection from chilling (C. Frenkel, personal communication), g, and 8.302 ± 2.015 g, respectively. and biogenesis of flavor and fragrance volatiles (Hatanaka et al., extraction from achenes. Protein extractions from 1973; Noueiry, 1989; Tress1 and Albrecht, 1986; Yamashita et al., achenes and receptacles were performed at 4C. Achenes were 1978). removed from ripe strawberries, washed to remove debris, and air Biosynthetic pathways of alcohols, aldehydes, and esters impli- dried. Achene tissue was ground in 1.5-ml microfuge tubes with cated in flavor and fragrance production have been tentatively tapered glass rods in an extraction buffer composed of 100 mM determined for many fruit (Tress1 and Albrecht, 1986). In straw- Tris-HCl, pH 7.5, 2% w/v soluble polyvinylpyrrolidone (PVP) as berries, exogenously applied fatty acids, aldehydes and alcohols a phenolic scavenger, 1 mM ZnSO4, 10 mM b- mercaptoethanol (Yamashita et al., 1977; Yamashita et al., 1979) were metabolized (BME) as a reducing agent, and the protease inhibitors 0.8 mM to their corresponding alcohols and esters. These results suggested phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine hy- that endogenous alcohols and aldehydes are interconverted by drochloride (BHC), and 5 mM e- amino-n-caproic acid (ACA). 100 ADH in the pathway for producing the complex mix of volatile µl of buffer was used per 10 mg of fresh weight. The extract was compounds (Honkanen and Hirvi, 1990) responsible for the char- centrifuged for 15 min at 13,600× g and the supernatant was acteristic flavor and fragrance of strawberries. collected as the enzyme source. The present work examines the substrate specificity of straw- Protein extraction from receptacles. Strawberries were har- berry ADH activities, the pattern of the NAD-dependent vested and achenes were removed from the receptacles by peeling ADH, and changes in ADH activities that occur during fruit the epidermis away with razor blades. Achene-free receptacles ripening and development. Our results support the hypothesis that were then homogenized in an extraction buffer composed of 100

NAD-dependent and NADP-dependent ADH enzymes are impor- mM Tris-HCl (pH 8.5), 2% w/v PVP, 20 mM CaCl2, 0.1 mM ZnSO4, tant components of the in vivo biosynthetic pathway of strawberry 0.8 mM PMSF, 1 mM BHC, 5 mM ACA, and 10 mM BME in a food flavor and fragrance volatiles. blender. The extract was filtered through one layer of Miracloth (Calbiochem) and the pH of the supernatant was adjusted to 7.5. The extract was then stirred for 15 min and centrifuged at 30,000× Received for publication 18 July 1994. Accepted for publication 10 Apr. 1995. This g to remove insoluble residues. The supernatant was made to 80% research was supported by grant no. 12-113 from the New Jersey Agricultural saturation in ammonium sulfate, stirred for 30 min, and centri- Experiment Station. We thank Chaim Frenkel and Joseph Goffreda for their critical fuged 20,000× g for 15 min to precipitate protein. The pellet was review of the manuscript. Use of trade names does not imply endorsement of the resuspended in a minimal volume of a buffer composed of 100 mM products named nor criticism of similar ones not named. The cost of publishing this Tris-HCl (pH 7.5), 0.1 mM ZnSO , 0.8 mM PMSF, 1 mM BHC, 5 mM paper was defrayed in part by the payment of page charges. Under postal regula- 4 tions, this paper therefore must be hereby marked advertisement solely to indicate ACA, and 10 mM BME and dialyzed against 50 mM Tris-Cl (pH this fact. 7.5), 0.1 mM ZnSO4, 0.8 mM PMSF, 1 mM BHC, 5 mM ACA, and 1 Present address: Biology Dept., Jersey City State College, Jersey City, NJ 07305. 10 mM BME.

798 J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. The protein extraction method for developmental studies was used when comparing relative activities for different substrates simpler. Achenes were removed from receptacles as above and the (Tables 2 and 3), except for the substrates cinnamyl alcohol (20 achene-free receptacles were homogenized in a Potter-Elvehjem mM) and geraniol (5 mM), which have relatively poor solubility in tissue grinder, in an extraction buffer composed of 100 mM Tris- aqueous solutions. Calculation of NAD(P) formed was assessed by

HCl (pH 8.5), 2% w/v PVP, 0.1 mM ZnSO4, 0.8 mM PMSF, 1 mM the change in A340 (Worthington, 1988). Preliminary results with

BHC, 5 mM ACA, and 10 mM BME. The extract was adjusted to pH strawberry NAD-dependent ADH gave a KM with as 7.5 and centrifuged at 13,600× g for 15 min to pellet insoluble substrate of 6.96 ± 0.32 mM, and strawberry NADP-dependent debris. The supernatant was used as the enzyme source. ADH gave a KM with benzyl alcohol of 24.82 ± 1.28 mM. Our KM ADH assays. All ADH assays were carried out at 24C. Oxida- values were similar to those of other plant NAD- and NADP- tions of alcohols by ADH were assayed in reaction mixes contain- dependent ADH enzymes (Bicsak et al., 1982; Davies et al., 1973; ing 100 mM glycine-KOH (pH 9.5) and 1.3 mM NAD or NADP, Roe et al, 1984; Yamashita et al., 1978). while reductions of aldehydes were assayed in reaction mixes Native isoelectric focusing. Native isoelectric focusing (IEF) containing 100 mM Tris-HCl (pH 7.5) with 1.3 mM NADH or was performed on a horizontal flat bed Fisher Biotech FN 1001 IEF NADPH. Substrate concentrations of 100 mM were used to devel- system according to manufacturers instructions. Gels 1 mm thick opmental course assays. Substrate concentrations of 40 mM were were composed of 1% (w/v) agarose, 13% (w/v) sorbitol, 6.7% (v/

Table 1. ADH activities of stage four strawberries.Z

“Activities were determined for primary fruit of genotype NJ 8343-6. NAD-ADH activity was measured using 100 mM ethanol as substrate, and NADP-ADH activity was measured using 100 mM benzyl alcohol as substrate.

Table 2. Substrate specificity of strawberry NAD-ADH.z

“Specific activity for NAD-ADH (genotype NJ 8336-l) was measured using 40 mM substrate except in the cases of cinnamyl alcohol and geraniol, where 20 mM and 5 mM were used, respectively. Values are ± SE.

Table 3. Substrate specificity of strawberry NADP-ADH.z

zSpecific activity for NADP-ADH (genotype NJ 8336-l) was measured using 40 mM substrate except in the cases of cinnamyl alcohol and geraniol, where 20 mM and 5 mM were used, respectively. Values are ± SE.

J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. 799 v) ampholines (pH 4 to 6.5), and 1% (v/v) Triton X-100 (Fisher). Isozyme differences may account for the differences in substrate

The electrode buffers were 0.05 N H2SO4 and 1 N NaOH. Electro- specificity we have seen between our achene NAD-dependent phoresis was carried out with 15 W constant power for 2 h at 9C. ADH and that of Yamashita et al. (1978) since the plants were After electrophoresis, gels were stained for NAD-dependent ADH genetically different. activity, with ethanol as substrate as by Noueiry (1989). Developmental control of ADH activity. The hypothesis that Staining IEF gels for NADP-ADH activity using benzyl alco- NAD-dependent and NADP-dependent ADH activities play im- hol as substrate was unsuccessful. Eggplant fruit and seed NADP- portant roles in flavor and fragrance production would be dis- dependent ADH enzymes were stainable with NADP and proved if ADH activities did not increase before or during the time various substrates (including benzyl alcohol and coniferyl alco- of fruit volatile production, that is, before or during the ripening hol) after nondenaturing polyacrylamide gel electrophoresis (un- process (stages 3 and 4). The ADH activities of entire strawberry published data). This leads us to believe that NADP-dependent receptacles were determined by enzyme assays for the four growth ADH is unstable during IEF. stages described in materials and methods. Total NAD-dependent The pI measurements of histochemically localized ADH ADH activity increased dramatically between stages 2 and 3 and isozymes were determined by directly measuring the pH gradient afterward remained at relatively high levels (Fig. 2), whereas total in the gels (Garlin, 1990). NADP-dependent ADH activity increased gradually from stage 2 to stage 4 (Fig. 3). Specific activities of strawberry receptacle Results and Discussion NAD- and NADP-dependent ADH (Figs. 2 and 3) showed their greatest increase between stages 3 and 4. Our hypothesis about ADH activities in fruit. NAD- and NADP-dependent ADH fruit ADH has therefore not been violated by the time course of activities of stage four strawberries are compared in Table 1. NAD- ADH inductions during fruit growth and ripening. Our results dependent ADH activity was 65.6-fold greater and NADP-depen- agree with the observed developmental increases of NAD-depen- dent ADH was 116-fold greater for achene tissue than for recep- dent ADH in oranges and tomatoes (Chen and Chase, 1993; Roe et tacle tissue on a fresh-weight basis. The total NAD-dependent al, 1984) and increases of the NAD- and NADP-dependent ADH ADH activity of achenes on a stage 4 fruit was 31.3% less than the of eggplants (unpublished data). activity found in receptacles, while the total NADP-dependent Functions of fruit ADH. The well-documented role of NAD- ADH activity of achenes was 2 1.2% greater than the activity found dependent ADH’s alleviating hypoxic stress through anaerobic in receptacles. NAD-dependent ADH activity was found to be 3.9- respiration (Freeling et al., 1985) and the role of some NADP- and 6.9-fold greater than the NADP-dependent activities found in dependent ADH enzymes (cinnamyl alcohol .dehydrogenases), achene and receptacle tissues respectively. which synthesize cinnamyl alcohols and thereby allow lignifica- The NAD-dependent ADH activity of receptacles (Table 2) was tion of woody tissues, seem to be inappropriate roles in strawber- highest against ethanol and showed considerable activity against ries. Strawberries have large surface to volume ratios, large inter- other unbranched primary alcohols. The receptacle enzyme also cellular air spaces and lack a thick cuticle (Darrow, 1966), charac- showed high activity against terpene (e.g., geraniol), branched teristics that allow free gas exchange and reduce the likelihood of (e.g. isoamyl alcohol), and unsaturated six-carbon (e.g., cis-3- fruit hypoxia; their vasculature is unlikely to require significant hexen-1-ol) alcohols. lignification. Strawberry receptacle NADP-dependent ADH (Table 3) showed A putative role for strawberry NADP-dependent ADH in chill- greatest activity against aromatic (e.g., cinnamyl alcohol and ing protection can not be ruled out. Work on a possible stimulatory benzyl alcohol) and terpene alcohols and a lesser activity against role for ADH in strawberry ripening (Mitchell and Jelenkovic, four- and six-carbon primary alcohols. A weak correlation existed 1992) remains inconclusive. between the hydrophobicity of each substrate and the activity of Alcohols and aldehydes contribute to the characteristic flavor the NADP-dependent enzyme for each substrate (e.g., the activity and fragrance of ripe fruit (Tress1 and Albrecht, 1986). The against aromatic alcohols and geraniol > four and six carbon alcohols that contribute to the flavor of ripe strawberries (Honkanen primary alcohols > ethanol). The reduction of an aliphatic and an aromatic by receptacle ADH was assessed. NAD-dependent ADH reduced 123465678 at twice the rate it reduced benzaldehyde, whereas, NADP-dependent ADH reduced benzaldehyde at nearly six times the rate of acetaldehyde. The specificity of the NAD-dependent ADH of achenes (Table 2) was similar to that found in receptacles, except that the achene enzyme showed less relative activity for terpene alcohols and more relative activity for unsaturated six carbon alcohols and aromatic alcohols. Achene NADP-dependent ADH (Table 3) showed lesser affinity for branched alcohols and higher affinity for secondary straight-chained alcohols compared to receptacle NADP-depen- dent ADH. Figure 1 shows a zymogram of strawberry achene and recep- tacle NAD-dependent ADH isozymes after separation by native IEF. Strawberry receptacles exhibited three to six isozymes de- Fig. 1. Native IEF of stage four strawberry fruit NAD-ADH isozymes. Protein from pending on the genotype. Noueiry (1989) had previously docu- 10 mg of achenes or 250 mg of receptacles were separated and histochemically developed on isoelectric focusing gels. The cathode is at the top of the figure. mented the presence of strawberry achene NAD-dependent ADH Lanes from left to right are 1) ‘Raritan’ achenes, 2) ‘Earliglow’ achenes, 3) NJ isozymes. Differences in isozymes might confer differences in the 8336-l achenes, 4) NJ 861 I-1 achenes, 5) ‘Raritan’ receptacles, 6) ‘Earliglow’ substrate specificities of the enzymes from different sources. receptacles, 7) NJ 8336-l receptacles, 8) NJ 861 l-l receptacles.

800 J. AMER. SOC. HORT: SCI. 120(5):798-801. 1995. Fig. 2. Developmental changes in NAD- dependent ADH activities in whole strawberry receptacles (genotype NJ 8336-1) over time. Assays used 100 mM ethanol as substrate as described under materials and methods. Bars represent the SE of the means. and Hit-vi, 1990) were good substrates for one or both of the ADH activities character- ized in this paper. In the future, we may be able to engineer strawberry flavor by select- ing for specific fruit ADH isozymes. Ge- netic transformation of strawberry plants with a previously isolated gene for NAD- dependent ADH (Wolyn and Jelenkovic, 1990) could change the ADH present in the fruit and thereby allow direct analysis of the effect of an added ADH isozyme on fruit flavor and fragrance. producing cis-3-hexenal and n-hexanol from linolenic and linoleic acids Our present work has indicated that 1) strawberry NAD- and in some plants. Phytochemistry 17:869-872. NADP-dependent ADH could use the kinds of alcohols and Honkanen, E. and Hirvi. 1990. The flavour of berries, p. 125-193. In: I.D. aldehydes found in ripe strawberries as substrates (NAD-depen- Morton and A.J. MacLeod (eds.). Food flavours. Part C. The flavour of dent ADH was more active against terpene and aromatic sub- Fruits. Elsevier, Amsterdam. Kanellis, A.K., T. Solomos, and K.A. Roubelakis-Angelakis. 1991. Sup- strates), 2) receptacle NAD- and NADP-dependent ADH substrate pression of cellulose and polygalacturonase isozymes in avocado fruit specificities differed slightly, from those of the achene, suggesting mesocarp subjected to low stress. Plant Physiol. 96:269-274. a difference in ADH isozymes between tissues involved in flavor Mitchell, W.C. and G. Jelenkovic. 1992. NAD-dependent and NADP- production (receptacle) and those not involved in flavor produc- dependent alcohol dehydrogenase enzymes: Analysis of their functional tion (achenes); 3) NAD-dependent ADH was composed of poly- significance in strawberry fruits. HortScience 27:594. (Abstr.) morphic isozymes as shown by native IEF; 4) isozyme differences Noueiry, A. 1989. Studies of differential activity and genetic nature of were seen between receptacle and achene tissues of the same alcohol dehydrogenase isozymes in various plant parts of the octoploid genotype, showing additional ADH isozymes in the flavor produc- strawberry, genotype 8343-6. MS thesis. Rutgers Univ. tion tissue (receptacle); and 5) the level of fruit ADH enzymes Roe, B., P.L. Davis, and J.H. Bruemmer. 1984. Pyruvate increased before and during ripening when they would be needed during maturation of Hamlin oranges. Phytochemistry 23:713-717. Tressl, R. and W. Albrecht. 1986. Biogenesis of aroma compounds for flavor and fragrance volatile production. These results strengthen through acyl pathways, p. 114-133. In: T.H. Parliment and R. Croteau the hypothesis that NAD- and NADP-dependent ADH enzymes (eds.). Biogeneration of aromas. Amer. Chem. Soc., Washington, D.C. function as important components of the in vivo biosynthetic Wolyn, D.J. and G. Jelenkovic. 1990. Nucleotide sequence of an alcohol machinery of strawberry flavor and fragrance volatiles. dehydrogenase gene in octoploid strawberry (Fragaria × ananassa Duch). Plant Mol. Biol. 14:855-857. Literature Cited Worthington, C.C. 1988. Worthington enzyme manual. Worthington Biochemical Corp., Freehold, N.J. Bicsak, T.A., L.R. Kann, A. Reiter, and T. Chase. 1982. Tomato alcohol Yamashita, I., K. Iino, Y. Nemoto, and S. Yoshikawa. 1977. Studies on flavor dehydrogenase: Purification and substrate specificity. Arch. Biochem. development in strawberries. 4. Biosynthesis of volatile alcohol and esters from Biophys. 216:605-615. aldehyde during ripening. J. Agr. Food Chem. 42: 1125-l 132. Chen, A.-R.S. and T. Chase. 1993. Alcohol dehydrogenase 2 and pyruvate Yamashita, I., K. Iino, and S. Yoshikawa. 1978. Alcohol dehydrogenase decarboxylase induction in ripening and hypoxic tomato fruit. Plant from strawberry seeds. Agr. Biol. Chem. 42:1125-l 132. Physiol. Biochem. 31:875-885. Yamashita, I., K. Iino, and S. Yoshikawa 1979. Substrate specificity in biosyn- Darrow. G.M. 1966. The Strawberry: history. breeding and physiology. thesis of volatile esters in strawberry fruit. Nip. Sho. Kog. Gak. 26:256-259. Holt, Rinehart and Winston, New-York. Davies, D.D., E.N. Ugochuku, K.D. Patil, and G.H.N. Towers. 1973. Aromatic alco- hol dehydrogenase from potato tubers. Phy- tochemistry 12:531-536. Freeling, M., and D.C. Bennett. 1985. Adh 1. Annu. Rev. Genet. 19:297-323. Garfin, D.E. 1990. Isoelectric focusing, p. 459- 477. In: M.P. Deutscher (ed.). Methods in enzymology. vol. 182. Guide to protein puri- fication. Academic Press, Sari Diego. Hatanaka, A., J. Sekiya, and T. Kajiwara. 1973. Distribution of an enzyme system

Fig. 3. Developmental changes in NADP- dependent ADH activities in whole strawberry receptacles (genotype NJ 8336-l) over time. Assays used 100 mM benzyl alcohol as substrate as described under materials and methods. Bars represent the SE of the means.

J. AMER. SOC. HORT. SCI. 120(5):798-801. 1995. 801